Journal: International Journal of Molecular Sciences

Article Title: MAD2L2 Dimerization Is Not Essential for Mitotic Regulation

doi: 10.3390/ijms252111485

Figure Lengend Snippet: Monomeric MAD2L2 can bind to CDH1: ( A ) Schematic representation of MAD2L2 residues mutated to disrupt homodimerization. ( B ) Myc-2DD and myc-3DD mutants displayed reduced binding to YFP-MAD2L2. HEK293 cells were co-transfected with the indicated plasmids, and α-myc IP was performed to assess MAD2L2 homodimer formation. The binding of myc-MAD2L2 to YFP-MAD2L2 was reduced in all myc-MAD2L2 mutants when compared to WT. **IgL–IgG Light chain. ( C ) MAD2L2-CDH1 binding was reduced in the HA-3DD mutant. HEK293 cells were co-transfected with the indicated plasmids, and α-myc IP was performed to assess MAD2L2-CDH1 binding. The binding of myc-CDH1 to HA-MAD2L2 was reduced in HA-MAD2L2 3DD and HA-R124A mutants when compared to WT and HA-2DD. ( D ) The quantification of the relative amount of myc CDH1 bound to each HA-MAD2L2 mutant. Error bar = 1 SD. The additional blots contributing to this analysis are shown in . ( E ) HA-MAD2L2 A135D mutant displayed reduced binding to mRFP-CDH1. HEK293 cells were co-transfected with the indicated plasmids, and α-HA IP was performed to assess MAD2L2-CDH1 binding. The binding of HA-MAD2L2 A135D mutant to mRFP-CDH1 was reduced compared to WT. ( F ) The alignment of the SHLD3 RFIP motif to the proposed CDH1 RFIP motif, revealing conserved residues shared in both proteins. ( G ) HA-CDH1 F48A exhibited binding to MAD2L2. HEK293 cells were co-transfected with the indicated plasmids, and α-GFP IP was performed to assess MAD2L2-CDH1 binding. The binding of both HA-CDH1 WT and F48A was detected. ( H ) HA-CDH1 F48A binding to endogenous CDC27 (APC3) was reduced. HEK293 cells were co-transfected with HA CDH1 WT or F48A mutant, and α-HA IP was performed to assess CDC27 CDH1 binding.

Article Snippet: Guide RNA sequences targeting MAD2L2 were phosphorylated, annealed, and ligated into a pX330-U6-Chimeric_BB-CBh-hSpCas9 vector (#42230, Addgene, a gift from Dr. Gabi Gerlitz Ariel University).

Techniques: Binding Assay, Transfection, Mutagenesis

Journal: International Journal of Molecular Sciences

Article Title: MAD2L2 Dimerization Is Not Essential for Mitotic Regulation

doi: 10.3390/ijms252111485

Figure Lengend Snippet: Monomeric MAD2L2 can regulate mitotic entry: ( A ) Clones of U2OS CRISPR/Cas9 knockout mad2l2 cell line and the stably complemented mad2l2 #1 cell line with different HA-tagged MAD2L2 mutants, as indicated. ( B ) The quantification of the time taken for knockout U2OS mad2l2 and the complemented HA- mad2l2 cells to reach the metaphase–anaphase transition from nuclear envelope breakdown (NEBD), assessed by time-lapse video microscopy (1 frame every 5 min). The plot shows the cumulative percentage of cells that progressed to anaphase. “n” represents the number of cells examined, collected from at least three independent experiments. The p -value between U2OS and mad2l2 cell lines was calculated using a one-tailed t -test. ( C ) Percentage of anaphase bridges (upper panel) and lagging chromosomes (lower panel) in mad2l2 and the complemented HA- mad2l2 U2OS cell lines. “n” represents the number of anaphases examined; error bar = 1 SD; n.s-not significant; p -value between the indicated cell lines was calculated using a one-tailed t -test.

Article Snippet: Guide RNA sequences targeting MAD2L2 were phosphorylated, annealed, and ligated into a pX330-U6-Chimeric_BB-CBh-hSpCas9 vector (#42230, Addgene, a gift from Dr. Gabi Gerlitz Ariel University).

Techniques: Clone Assay, CRISPR, Knock-Out, Stable Transfection, Microscopy, One-tailed Test

Journal: International Journal of Molecular Sciences

Article Title: MAD2L2 Dimerization Is Not Essential for Mitotic Regulation

doi: 10.3390/ijms252111485

Figure Lengend Snippet: Monomeric MAD2L2 inhibits APC/C activation: ( A ) mad2l2 clones present premature binding of CDH1 to the APC/C. In order to assess the binding of CDH1 to the APC/C in each cell line, an α-CDC27 IP was performed on U2OS and mad2l2 clones that were arrested in nocodazole for 16h. In both mad2l2 clones, but not in WT cells, CDH1 prematurely binds to the CDC27 subunit of the APC/C, during nocodazol arrest. ( B ) mad2l2 :HA-MAD2L2 restores the canonical binding pattern of CDH1 to the APC/C and prevents premature binding of CDH1 to the APC/C. In order to assess the binding of CDH1 to the APC/C in each cell line, an α-CDC27 IP was performed on cells arrested in nocodazole for 16h. WT and mad2l2 :HA-MAD2L2 cells present lower binding to the CDC27 subunit, compared to mad2l2 cells. ( C ) mad2l2 :HA-MAD2L2 2DD is able to prevent the premature binding of CDH1 to the APC/C. In order to assess the binding of CDH1 to the APC/C in each cell line, an α-CDC27 IP was performed on cells arrested in nocodazole for 16 h. WT and mad2l2 :HA-MAD2L2 2DD present lower binding present lower binding to the CDC27 subunit, compared to mad2l2 cells. ( D ) Both mad2l2 :HA-MAD2L2 3DD and mad2l2 :HA-MAD2L2 A135D present premature binding of CDH1 to the APC/C and fail to restore the normal binding pattern, as indicated by the relatively high level of CDH1 bound to CDC27 during nocodazole treatment. **IgL–IgG Light chain. ( E ) The quantification of the relative amount of CDH1 bound to CDC27 in all presented cell lines. Each dot represents an independent repeat of the IP. The blots contributing to this analysis are shown in –E. ( F ) Premature binding of CDH1 to the APC/C, during nocodazole arrest, leads to a reduction in CDH1 levels. Endogenous levels of CDH1 were monitored in the indicated cell lines after 16 h of nocodazole treatment. Lack of MAD2L2, or the presence of MAD2L2 without the ability to bind CDH1, leads to premature CDH1 degradation. ( G ) The quantification of the relative amount of endogenous CDH1 in nocodazole-arrested cells. Each dot represents an independent repeat. Blots contributing to this analysis are shown in . ( H ) Premature binding of CDH1 to the APC/C, during nocodazole arrest, leads to a reduction in Aurora A levels. Endogenous levels of Aurora A were monitored in the indicated cell lines after 16h of nocodazole treatment. Lack of MAD2L2, or the presence of MAD2L2 without the ability to bind CDH1, leads to premature Aurora A degradation. ( I ) The quantification of the relative amount of endogenous Aurora A in nocodazole-arrested cells. Each dot represents an independent repeat. The additional blots contributing to this analysis are shown in . For all: Error bar = 1 SD; p -value was calculated using a one-tailed t -test.

Article Snippet: Guide RNA sequences targeting MAD2L2 were phosphorylated, annealed, and ligated into a pX330-U6-Chimeric_BB-CBh-hSpCas9 vector (#42230, Addgene, a gift from Dr. Gabi Gerlitz Ariel University).

Techniques: Activation Assay, Clone Assay, Binding Assay, One-tailed Test

Journal: International Journal of Molecular Sciences

Article Title: MAD2L2 Dimerization Is Not Essential for Mitotic Regulation

doi: 10.3390/ijms252111485

Figure Lengend Snippet: CDH1 overexpression reduces MAD2L2 homodimerization: ( A ) A graphic model presenting two options for MAD2L2-CDH1 interaction. Option 1: CDH1 can bind both monomeric and dimeric forms of MAD2L2 via the L186 residue. Option 2: CDH1 binds to the monomeric form of MAD2L2 using both L186 and A135 residues, blocking MAD2L2 homodimerization. ( B ) In situ MAD2L2 homodimer formation is reduced upon CDH1-GFP overexpression, as indicated by a reduced number of foci/cell. Representative images of the PLA foci. ( C ) The quantification of MAD2L2 homodimer from the PLA assay; error bar = 1 SD; each dot represents a cell. ( D ) The number of foci/cell for each cell line in a cumulative percentage plot to observe the difference between cell populations upon treatments; p -value was calculated using a one-tailed t -test.

Article Snippet: Guide RNA sequences targeting MAD2L2 were phosphorylated, annealed, and ligated into a pX330-U6-Chimeric_BB-CBh-hSpCas9 vector (#42230, Addgene, a gift from Dr. Gabi Gerlitz Ariel University).

Techniques: Over Expression, Residue, Blocking Assay, In Situ, One-tailed Test

Journal: The EMBO Journal

Article Title: Synergistic and antagonistic activities of IRF8 and FOS enhancer pairs during an immune-cell fate switch

doi: 10.1038/s44318-025-00380-w

Figure Lengend Snippet: Reagents and tools table

Article Snippet: The base px330 plasmid was a gift from Jinsong Li (Addgene plasmid # 98750; http://n2t.net/addgene:98750 ; RRID: Addgene_98750 ).

Techniques: Recombinant, Clone Assay, Binding Assay, Sequencing, CRISPR, Magnetic Beads, Software, Nick Translation, Electron Microscopy, Plasmid Preparation, DNA Extraction, Reverse Transcription, Purification

Journal: Nature Communications

Article Title: SERCA2 phosphorylation at serine 663 is a key regulator of Ca 2+ homeostasis in heart diseases

doi: 10.1038/s41467-023-39027-x

Figure Lengend Snippet: A Representative curves of normalized [Ca 2+ ] ER ratio signals over time measured with the genetically encoded Ca 2+ probe D1ER in HEK293-T cells in Ca 2+ free and 1 mM EGTA-containing buffer for three minutes before depleting the ER Ca 2+ store by adding 100 µM cyclopiazonic acid (CPA) and 500 µM ATP-Na 2+ for seven minutes and prior to ER Ca 2+ refilling by switching for six minutes and a half to 2 mM Ca 2+ , 500 µM ATP-Mg 2+ and 1 µM ionomycin-containing buffer (green curve), or supplemented with 10 µM TDZD8 (orange curve), or 1 µM thapsigargin (gray curve) to compare SERCA activity. Ratio signals were normalized as following: [1+ ( R − R t =600s )/(R t=180s. − R t=600s )]. B Violin plot of ER Ca 2+ refilling slope over two minutes from time 630 s of control ( n = 199, on 11 experimental days), TDZD8 ( n = 210, on 7 experimental days), thapsigargin ( n = 42, on 2 experimental days) and 1 µM SB216763 (N = 212 cells, on 6 experimental days). Median with a 95% confidence interval is shown and one-way ANOVA followed by Tukey’s multiple comparisons test was used to assess significance versus Control condition (**** p < 0.0001). C Motif of the GSK3β substrate sequence (from PhosphoSitePlus database). D Listing of in silico analysis of putative GSK3β target sites on SERCA2 determined by NetPhos3.1 and PhosphoSitePlus public databases. E Mass spectrometry analysis of SERCA2 phosphorylation at serine 663 residue (Sp663) in left ventricle of C57BL/6 mice in basal (Control; n = 10) and after 60 min ischemia followed by 30 minutes reoxygenation (IR; n = 10). Median with interquartile range is shown and two-tailed Mann–Whitney test was used to assess significance (* p = 0.027). F Mass spectrometry analysis of SERCA2 phosphorylation at serine 663 in healthy patients (Non-failing; n = 5) and patient with end-stage heart failure (Failing; n = 5 regions). Median with interquartile range is shown and two-tailed Mann–Whitney test was used to assess significance (* p = 0.0159). G 3D structure of SERCA2, with α-helix and β-sheet in different SERCA2 domains. Visualization of the accessibility of the cytosolic phosphorylation site at serine 663 (SER663, yellow) at the periphery of the phosphorylation domain of SERCA2 (brown). Figure adapted from . H Interaction of GSK3β with SERCA2 determined by co-immunoprecipitation on lysates from HEK293-T cells (basal ( n = 3) and after 1 h hypoxia followed by 30 min reoxygenation ( n = 3)). GAPDH was used as a negative control. I Representative images of FRET between SERCA2-mTurquoise2 donor and GSK3β-sYFP2 acceptor, on living HEK293-T cells, in normoxic condition or after 60 min hypoxia followed by 30 minutes reoxygenation (H/R). Scale bar represents 10μm (left). Dot plot of the FRET efficiency in normoxia ( n = 15) and after H/R ( n = 24, *** p = 0.0009), on four independent experiments (right). Median with interquartile range is shown and two-tailed Mann–Whitney test was used to assess significance. J Representative confocal microscopy images of the in situ SERCA2-GSK3β proximity depicted as red dots on adult cardiomyocytes, in normoxic or after 45 minutes hypoxia followed by 120 min reoxygenation (H/R). Nuclei appear in blue (top). Quantification of the interactions per cell of normoxia ( N = 16) and H/R ( N = 32) groups, presented as a fold of Normoxia, on three independent experiments. Scale bar represents 10 μm (bottom). Median with interquartile range is shown and two-tailed Mann–Whitney test was used to assess significance (**** p < 0.0001).

Article Snippet: For substitution of serine 663 residue (TCC codon) of SERCA2 protein into phosphoresistant alanine (GCG codon), a gRNA was designed to anneal sense strand downstream targeted locus in the ATP2A2 ( SERCA2 ; ENSG00000174437) gene (base-pairing sequence: 5′.GTTCAGGCAGGCGTCTCGCTggg---.3′) and cloned into pX330S-2 (Addgene #58778), expressing both Cas9-nuclease and gRNA, according to the protocol defined by Sakuma et al. .

Techniques: Activity Assay, Control, Sequencing, In Silico, Mass Spectrometry, Phospho-proteomics, Residue, Two Tailed Test, MANN-WHITNEY, Immunoprecipitation, Negative Control, Confocal Microscopy, In Situ

Journal: Nature Communications

Article Title: SERCA2 phosphorylation at serine 663 is a key regulator of Ca 2+ homeostasis in heart diseases

doi: 10.1038/s41467-023-39027-x

Figure Lengend Snippet: A Representative curves of normalized [Ca 2+ ] ER ratio signals over time measured with the genetically encoded Ca 2+ probe D1ER in HEK293-T wild-type (green curve), Serca2[S663A] phosphoresistant mutant (red curve) and Serca2[S663A] phosphoresistant mutant supplemented with 10 µM TDZD8 for ER Ca 2+ refilling (dashed orange curve). Ratio signals were normalized as following: [1 + ( R − R t =600s )/( R t=180sec. − R t=600s )]. B Violin plot of the ER Ca 2+ refilling slope over two minutes from time 630 s of WT ( N = 108 cells on 4 experimental days), Serca2[S663A] ( N = 108 cells on 9 experimental days) and Serca2[S663A] + TDZD8 ( N = 100 cells on 4 experimental days) respectively. Median with a 95% confidence interval is shown and one-way ANOVA followed by Tukey’s multiple comparisons test was used to assess significance (**** p < 0.0001) (ns p = 0.3953, **** p < 0.0001). C Violin plot of basal [Ca 2+ ] ER (endoplasmic reticulum) ratio measured with D1ER probe in HEK293-T wild-type (WT) ( N = 117) and Serca2[S663A] phosphoresistant mutant cells ( N = 108). Median with distribution is shown and two-tailed unpaired t test with Welch’s correction was used to assess significance (**** p < 0.0001). D Violin plot of basal [Ca 2+ ] CYTO (cytosol) ratio measured with D3cpV probe in HEK293-T wild-type (WT) ( N = 214) and Serca2[S663A] phosphoresistant mutant cells ( N = 124). Median with distribution is shown and two-tailed unpaired t test with Welch’s correction was used to assess significance (**** p < 0.0001). E Violin plot of basal [Ca 2+ ] MITO (mitochondria) ratio measured with 4mtD3cpV probe, in HEK293-T wild-type (WT) ( N = 219) and Serca2[S663A] phosphoresistant mutant cells ( N = 197). Median with distribution is shown and two-tailed unpaired t test with Welch’s correction was used to assess significance (**** p < 0.0001). F Representative curves of normalized [Ca 2+ ] ER ratio signals over time measured with the genetically encoded Ca 2+ probe D4ER in human induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CM) infected (6 days, MOI 100 000) with the AAV9-Serca[WT], wild-type (green curve), the AAV9-Serca2[S663A] phosphoresistant mutant (red curve) and the AAV9-Serca2[S663E] phosphomimetic mutant (blue curve). G Violin plot of ER Ca 2+ refilling slope over four minutes from time 1119 s in AAV9-Serca[WT] ( N = 44 cells on 4 experimental days), AAV9-Serca2[S663A] ( N = 19 cells on 4 experimental days) and AAV9-Serca2[S663E] ( N = 35 cells on 4 experimental days) infected hiPSC-CM cells. Median with a 95% confidence interval is shown and one-way ANOVA followed by Tukey’s multiple comparisons test was used to assess significance (ns p = 0.2212, * p = 0.0296, *** p = 0.0007). H Violin plot of basal [Ca 2+ ] ER (endoplasmic reticulum) ratio signal measured with D4ER probe (6 days, MOI 100 000) in hiPSC-CM infected with AAV9-Serca2[WT] ( N = 57 on 4 experimental days), -Serca2[S663A] ( N = 19 on 4 experimental days)or -Serca2[S663E] ( N = 64 on 4 experimental days). Median with a 95% confidence interval is shown and one-way ANOVA followed by Tukey’s multiple comparisons test was used to assess significance (* p = 0.0115, **** p < 0.0001).

Article Snippet: For substitution of serine 663 residue (TCC codon) of SERCA2 protein into phosphoresistant alanine (GCG codon), a gRNA was designed to anneal sense strand downstream targeted locus in the ATP2A2 ( SERCA2 ; ENSG00000174437) gene (base-pairing sequence: 5′.GTTCAGGCAGGCGTCTCGCTggg---.3′) and cloned into pX330S-2 (Addgene #58778), expressing both Cas9-nuclease and gRNA, according to the protocol defined by Sakuma et al. .

Techniques: Mutagenesis, Two Tailed Test, Derivative Assay, Infection

Journal: Nature Communications

Article Title: SERCA2 phosphorylation at serine 663 is a key regulator of Ca 2+ homeostasis in heart diseases

doi: 10.1038/s41467-023-39027-x

Figure Lengend Snippet: A Cell death evaluated by flow cytometry with propidium iodide (PI) in normoxic and hypoxic WT ( N = 11) and Serca2[S663A] ( N = 12) groups. Cyclosporin A (CsA) ( N = 11) was used as a positive control. Median with a 95% confidence interval from 4 independent days is shown and two-way ANOVA followed by Tukey’s multiple comparisons test versus WT condition was used to assess significance (ns p ≥ 0.05, * p = 0.0119, *** p = 0.004. B Kinetic curves of [Ca 2+ ] ER (endoplasmic reticulum) ratio measured with D1ER probe at 0.5 h/1 h/2 h reoxygenation of HEK293-T wild-type (WT) (dotted green curve, N = 103/86/89 for respective reperfusion time, on 4 experimental days) and Serca2[S663A] phosphoresistant mutant (continuous red curve, N = 90/95/95 for respective reperfusion time, on 4 experimental days) cells exposed to 18 h hypoxia (1%). Mean with a 95% confidence interval is shown and two-tailed unpaired t test with Welch’s correction was used to assess significance (**** p < 0.0001). C Kinetic curves of [Ca 2+ ] CYTO (cytosol) ratio measured with D3cpV probe at 0.5 h/1 h/2 h reoxygenation of HEK293-T wild-type (WT) (dotted green curve, N = 71/44/36 for respective reperfusion time, on 4 experimental days) and Serca2[S663A] phosphoresistant mutant (continuous red curve, N = 64/73/72 for respective reperfusion time, on 4 experimental days) cells exposed to 18 h hypoxia (1%). Mean with a 95% confidence interval is shown and two-tailed unpaired t test with Welch’s correction was used to assess significance (**** p < 0.0001). D Kinetic curves of [Ca 2+ ] MITO (mitochondria) ratio measured with 4mtD3cpV probe at 0.5 h/1 h/2 h reoxygenation of HEK293-T wild-type (WT) (dotted green curve, N = 73/55/47 for respective reperfusion time, on 4 experimental days) and Serca2[S663A] phosphoresistant mutant (continuous red curve, N = 72/70/97 for respective reperfusion time, on 4 experimental days) cells exposed to 18 h hypoxia (1%). Mean with a 95% confidence interval is shown and two-tailed unpaired t test with Welch’s correction was used to assess significance (*** p = 0.009, **** p < 0.0001). E Cell death evaluated by flow cytometry with propidium iodide (PI) in hypoxic MEF-T Serca2 KO cell line, rescued with Serca2 wild-type (rWT), [S663A] phosphoresistant mutant (rS663A) and [S663E] phosphomimetic mutant (rS663E). Mean with a 95% confidence interval from n = 9 experiments on 3 independent days is shown and two-way ANOVA followed by Tukey’s multiple comparisons test versus rWT condition was used to assess significance (ns p = 0.8029, **** p < 0.0001). F Representative curves of normalized [Ca 2+ ] CYTO (cytosol) ratio signals over time measured with D3cpV probe in Serca2 null MEF-T cells and rescued with Serca2 wild-type (rWT; green curve), [S663A] phosphoresistant mutant (rS663A; red curve) or [S663E] phosphomimetic mutant (rS663E; blue curve) cells in Ca 2+ free and 1 mM EGTA-containing buffer for one minutes prior to add 100 µM ATP-Na 2+ . G Violin plots of normalized [Ca 2+ ] maximal increase after ATP-Na 2+ stimulation of rWT ( N = 22 cells on 3 independent days), rS663A ( N = 23 cells on 3 independent days) and rS663E ( N = 18 cells on 3 independent days). Median with a 95% confidence interval is shown and one-way ANOVA followed by Tukey’s multiple comparisons test was used to assess significance (**** p < 0.0001). H Cell death evaluated by flow cytometry with propidium iodide (PI) after H/R in hiPSC-CM cells infected with SERCA2 wild-type [WT] ( N = 7) or phosphoresistant [S663A] mutant ( N = 7). Median with interquartile range, from n = 7 independent experiments, is shown and two-tailed paired Wilcoxon test was used to assess significance (* p = 0.0313).

Article Snippet: For substitution of serine 663 residue (TCC codon) of SERCA2 protein into phosphoresistant alanine (GCG codon), a gRNA was designed to anneal sense strand downstream targeted locus in the ATP2A2 ( SERCA2 ; ENSG00000174437) gene (base-pairing sequence: 5′.GTTCAGGCAGGCGTCTCGCTggg---.3′) and cloned into pX330S-2 (Addgene #58778), expressing both Cas9-nuclease and gRNA, according to the protocol defined by Sakuma et al. .

Techniques: Flow Cytometry, Positive Control, Mutagenesis, Two Tailed Test, Infection

Journal: Nature Communications

Article Title: SERCA2 phosphorylation at serine 663 is a key regulator of Ca 2+ homeostasis in heart diseases

doi: 10.1038/s41467-023-39027-x

Figure Lengend Snippet: A Experimental design for in vivo gene therapy experiments with heart transverse section after TTC staining. Scale bar represents 500 μm. B Quantification of the area at risk (AR) expressed as percentage of left ventricle (LV) of SERCA2-KD mice rescued with SERCA2 -WT ( N = 13), -S663A ( N = 11) or -S663E ( N = 14). Mean with a 95% confidence interval is shown and one-way ANOVA followed by Tukey’s multiple comparisons test was used to assess significance (ns p ≥ 0.05). C Scatterplot of AN over the AR of mice rescued with SERCA2 -WT ( N = 13), -S663A ( N = 11) or -S663E ( N = 14). D Quantification of infarct size (AN) expressed as percentage of AR of mice rescued with SERCA2 -WT ( N = 13), -S663A (N = 11) or -S663E ( N = 14). Mean with a 95% confidence interval is shown and one-way ANOVA followed by Tukey’s multiple comparisons test was used to assess significance (* p = 0.0292 rWT vs rS663A, * p = 0.0144 rWT vs rS663E, **** p < 0.0001). E Dot plot of fitted linear regression slope of AN/AR of rWT ( N = 13), rS663A ( N = 11) or rS663E ( N = 14) SERCA2 rescued mice. Mean with a 95% confidence interval is shown and one-way ANOVA followed by Tukey’s multiple comparisons test was used to assess significance (* p = 0.0272 rWT vs rS663A, * p = 0.0136 rWT vs rS663E, **** p < 0.0001). F – I Evaluation of cardioprotective signaling pathways in lysates of cardiac area at risks. F Quantification of western blotting for Phospho-ERK over total ERK1-2 in rWT ( N = 11), rS663A ( N = 11) and rS663E ( N = 11) SERCA2 rescued mice. Mean with a 95% confidence interval, from n = 3 independent experiments, is shown and one-way ANOVA followed by Tukey’s multiple comparisons test was used to assess significance (ns p > 0.05). G Quantification of western blotting for phospho-STAT3 over total STAT3 in rWT ( N = 8), rS663A ( N = 8) and rS663E ( N = 8) SERCA2 rescued mice. Mean with a 95% confidence interval, from n = 3 independent experiments, is shown and one-way ANOVA followed by Tukey’s multiple comparisons test was used to assess significance (ns p > 0.05) H Quantification of western blotting for phospho S16-T17-PLN over total PLN, expressed as fold of rWT. Mean with a 95% confidence interval, from n = 3 independent experiments, is shown and one-way ANOVA followed by Tukey’s multiple comparisons test was used to assess significance (ns p = 0.8195, * p = 0.0102 rS663A vs rS663E, * p = 0.0424 rWT vs rS663E). I Quantification of the interaction of PLN with SERCA2 determined by co-immunoprecipitation on lysates from cardiac area at risks in rWT ( N = 6), rS663A ( N = 6) and rS663E ( N = 6) SERCA2 rescued mice. Mean with a 95% confidence interval, from n = 3 independent experiments, is shown and one-way ANOVA followed by Tukey’s multiple comparisons test was used to assess significance (ns p > 0.05). J – L Evaluation of the functions of the mouse cardiomyocytes isolated from the AR after the in vivo ischemia-reperfusion insult. J Cell death evaluated by flow cytometry with propidium iodide (PI) in rWT ( N = 7) and rS663A ( N = 7) SERCA2 rescued mice. Median with interquartile interval is shown and two-tailed unpaired t test with Welch’s correction was used to assess significance (* p = 0.0104). K Mitochondrial membrane potential evaluated with TMRM in rWT ( N = 7) and rS663A ( N = 8) SERCA2 rescued mice. Median with interquartile interval is shown and two-tailed unpaired t test with Welch’s correction was used to assess significance (*** p = 0.0002). L Resting cytosol Ca 2+ evaluated with the ratiometric FuraRed sensor in rWT ( N = 6) and rS663A ( N = 8) SERCA2 rescued mice. Median with interquartile interval is shown and two-tailed unpaired t test with Welch’s correction was used to assess significance (* p = 0.0105). M – P Quantification of sarcomere shortening in mouse cardiomyocytes isolated from the AR after the in vivo ischemia-reperfusion insult measured with the software IonWizard; Ionoptix system. M Evaluation of peak h in µm in rWT ( N = 135) and rS663A ( N = 245) SERCA2 rescued cardiomyocytes. Median with interquartile interval is shown and two-tailed unpaired t test with Welch’s correction was used to assess significance (**** p < 0.0001). N Evaluation of time to peak 50% in rWT ( N = 135) and rS663A ( N = 245) SERCA2 rescued cardiomyocytes. Median with interquartile interval is shown and two-tailed unpaired t test with Welch’s correction was used to assess significance (**** p < 0.0001). O Evaluation of time to baseline 50% in rWT ( N = 133) and rS663A ( N = 244) SERCA2 rescued cardiomyocytes. Median with interquartile interval is shown and two-tailed unpaired t test with Welch’s correction was used to assess significance (*** p = 0.0003). P Evaluation of area under curve in rWT ( N = 135) and rS663A ( N = 242) SERCA2 rescued cardiomyocytes. Median with interquartile interval is shown and two-tailed unpaired t test with Welch’s correction was used to assess significance (**** p < 0.0001).

Article Snippet: For substitution of serine 663 residue (TCC codon) of SERCA2 protein into phosphoresistant alanine (GCG codon), a gRNA was designed to anneal sense strand downstream targeted locus in the ATP2A2 ( SERCA2 ; ENSG00000174437) gene (base-pairing sequence: 5′.GTTCAGGCAGGCGTCTCGCTggg---.3′) and cloned into pX330S-2 (Addgene #58778), expressing both Cas9-nuclease and gRNA, according to the protocol defined by Sakuma et al. .

Techniques: In Vivo, Staining, Protein-Protein interactions, Western Blot, Immunoprecipitation, Isolation, Flow Cytometry, Two Tailed Test, Membrane, Software

Journal: Nature Communications

Article Title: SERCA2 phosphorylation at serine 663 is a key regulator of Ca 2+ homeostasis in heart diseases

doi: 10.1038/s41467-023-39027-x

Figure Lengend Snippet: In healthy heart, during excitation-contraction coupling (ECC) induced after depolarization of the sarcolemma, a small amount of Ca 2+ enters to the sarcoplasm through the voltage sensitive L-type calcium dihydropyridine channels (LTCC) of the T-tubule, targeting a rapid and large amount of Ca 2+ release (systolic Ca 2+ ) from inside the sarcoplasmic reticulum (SR/ER) through the Ca 2+ ryanodine channels (RyR), which subsequently activates myofilaments (MF) for muscle contraction. Relaxation occurs when SR Ca 2+ ATPase (SERCA2) reuptakes Ca 2+ , which is regulated by the balance of the phosphorylation level of SERCA2 at serine 663 (S663) via the GSK3β, lowering cytosolic Ca 2+ concentration in combination with Ca 2+ extrusion via Na + –Ca 2+ exchanger (NCX) working in reverse mode. Mitochondria (Mito) also participate taking and extruding Ca 2+ from the cytosol during Ca 2+ cycle. During ischemic heart disease, the level of SERCA2 phosphorylation at S663 is increased, reducing the reuptake of Ca 2+ into SR/ER during the relaxation phase, which contributes to the increase of both intracellular (Ca 2+ i ) and mitochondrial Ca 2+ (Ca 2+ m ) overload, driving cells towards death. Conversely, preventing SERCA2 phosphorylation at reperfusion, enhances the Ca 2+ reuptake into SR/ER, increasing the SR/ER Ca 2+ content, which subsequently (1) reduces the intracellular and mitochondrial Ca 2+ content, and (2) improves excitation-contraction coupling of cardiomyocytes, contributing to the protection and recovery of ischemic heart.

Article Snippet: For substitution of serine 663 residue (TCC codon) of SERCA2 protein into phosphoresistant alanine (GCG codon), a gRNA was designed to anneal sense strand downstream targeted locus in the ATP2A2 ( SERCA2 ; ENSG00000174437) gene (base-pairing sequence: 5′.GTTCAGGCAGGCGTCTCGCTggg---.3′) and cloned into pX330S-2 (Addgene #58778), expressing both Cas9-nuclease and gRNA, according to the protocol defined by Sakuma et al. .

Techniques: Phospho-proteomics, Concentration Assay

Journal: Oncogenesis

Article Title: Androgen receptor variant 7 exacerbates hepatocarcinogenesis in a c-MYC-driven mouse HCC model

doi: 10.1038/s41389-023-00449-3

Figure Lengend Snippet: A RNA-seq transcriptome analysis showed that 150 genes that were upregulated by c-MYC were repressed by AR-FL and/or AR-V7 co-expression; and 31 genes that were downregulated by c-MYC were upregulated by AR-FL and/or AR-V7 co-expression in both male (left) and female (right) mice at 20 dpi respectively. Each blue line represents differential expression levels (fold change in reference to control, Y axis) in the tumors of the respective combination of injected gene(s), X axis. Red lines highlight the differential expression levels of Cldn7 in respective samples. B Quantitative RT-PCR for expressions of Cldn7 in respective groups at 20 dpi. Expression of Cldn7 in male mice of c-MYC/p53KO (hep-c-MYC/p53KO) tumor at 11 dpi (pink bar), indicating that Cldn7 was repressed in p53-deficient conditions and hence was likely regulated by p53 [70–72]. C Expression of p53 in respective mouse groups in male and female, indicating p53 was upregulated by c-MYC but not significantly affected by AR-FL or AR-V7 co-expression. D Immunofluorescent images of c-MYC (red), Cldn7 FLAG (green), and DNA (blue) in the liver of hep-c-MYC/p53KO/Cldn7 FLAG mouse at 2 dpi, showing co-expression of the respective transgenes in the same cells. Scale bar = 50 µm E , Macroscopic phenotypes of the livers in male mice at 11 dpi injected with the expression vectors of c-MYC (c-MYC) ( n = 5), c-MYC under p53-deficient condition (c-MYC/p53KO) ( n = 6), or c-MYC and Cldn7 FLAG under p53-deficient condition (c-MYC/p53KO/Cldn7 FLAG ) ( n = 5). F Liver to body weight ratio of the samples presented in E , indicating that the exacerbation of c-MYC-driven HCC under p53-deficient conditions was completely reversed by co-expression of Cldn7 FLAG . Statistical significance by one-way ANOVA with Tukey’s multiple comparisons test for B , C , and F ; * p < 0.05; ** p < 0.001; *** p < 0.0001. Error bars indicate mean ± SEM.

Article Snippet: The pT3-based c-MYC expression vector pT3-c-MYC (#92046) [ ], the SB100 transposase expression vector pCMV-SB100 (#34879) [ ], the CRISPR/Cas9-base mouse p53 knockout vector pX330-p53 (#59910) [ ], and the full-length human androgen receptor vector pLENTI6.3/AR-GC-E2325 (#85128) were obtained from the Addgene Repository (Watertown, MA).

Techniques: RNA Sequencing, Expressing, Quantitative Proteomics, Control, Injection, Quantitative RT-PCR

Journal: Oncogenesis

Article Title: Androgen receptor variant 7 exacerbates hepatocarcinogenesis in a c-MYC-driven mouse HCC model

doi: 10.1038/s41389-023-00449-3

Figure Lengend Snippet: AR-V7 potentiates the cancer-related genes and oncogenic processes regulated by c-MYC, while it represses a tumor suppressor Cldn7 that is upregulated by c-MYC-overexpression via its activation of the p53 signaling pathway. AR-FL partly exerts similar functions to those of AR-V7 in the presence of androgen in males.

Article Snippet: The pT3-based c-MYC expression vector pT3-c-MYC (#92046) [ ], the SB100 transposase expression vector pCMV-SB100 (#34879) [ ], the CRISPR/Cas9-base mouse p53 knockout vector pX330-p53 (#59910) [ ], and the full-length human androgen receptor vector pLENTI6.3/AR-GC-E2325 (#85128) were obtained from the Addgene Repository (Watertown, MA).

Techniques: Over Expression, Activation Assay